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2014 “ Towards an HIV Cure ” symposium Melbourne

2014 “ Towards an HIV Cure ” symposium Melbourne. Following in vitro culture with myeloid dendritic cells, negative regulators of T cell activation are expressed preferentially on latently infected CD4 + T cells

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2014 “ Towards an HIV Cure ” symposium Melbourne

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  1. 2014 “Towards an HIV Cure” symposiumMelbourne Following in vitro culture with myeloid dendritic cells, negative regulators of T cell activation are expressed preferentially on latently infected CD4+ T cells VanessaA. Evans, Renée M. van der Sluis, Nitasha A. Kumar, Rafick-Pierre Sekaly, Remi Fromentin, Nicolas Chomont, Paul U. Cameron, Sharon R. Lewin

  2. Resting CD4+T cell Infection of resting CD4+ T cells: a role for cell-cell interactions Unactivated resting cells Ex vivo tissue blocks Eckstein et al. J Virology 2001 Endothelial Cells Shenet al. J Virology 2013 Dendritic Cells Evans et al. PLoSPathogens 2013

  3. Myeloid DC induce latency in resting memory CD4+ T cells Non-proliferating CD4+ T cells • Not mediated by soluble factors: CCL19, CCL21, CXCL10, IL-10, IL-6 • Close DC-T cell proximity required for induction of latency Evans et al. PLoS Pathogens 2013

  4. Negative regulators and latency • Negative regulators dampen the immune response and can be found on exhausted T cells Ahmed et al. J Immunol 2010; Day et al. Nature 2006 • Latently infected T cells express negative regulators of T cell activation eg. PD-1, Tim-3 and TIGIT Chomont et al. Nat Med 2010; Fromentin et al. CROI 2014 • Blocking PD-L1/PD-1 in vivo restores the SIV-specific cellular and humoral immune responses, improves viral control and reduces immune activation Velu et al. Nature 2009; Dyavar Shetty et al. J Clin Invest 2012; Finnefrock et al. J Immunol 2009

  5. Hypothesis Expression of negative regulators during DC-T cell interactions may actively suppress viral replication and maintain latency

  6. Resting CD4+T cells Magnetic Bead Depletion Resting CD4+ T cells >98% PBMC CD4 + mouse anti-human CD8, CD11b, CD14, CD16, CD19, CD69, HLA-DR CD69 CD25 HLA-DR CD3 Dendritic Cells Magnetic Bead Depletion Plasmacytoid DC Bulk DC PBMC CD123 + mouse anti-human CD3, CD11b, CD19 HLA-DR Myeloid DC Lineage Cocktail CD11c

  7. DC added 1:10 AND 1 day Not productively infected EGFP- Non-proliferating eFluor670hi R5-EGFP-HIV-1 (2h pulse) Latent infection Resting CD4+ T cells eFluor670 Day 5 p.i. eFluor670hiEGFP- CD4+ T cells + integrase inhibitor L8 aCD3/aCD28 eFluor670 Activation >99% 3 Days EGFP Productive infection

  8. OR + mDC 1 day R5-EGFP-HIV-1 (2h pulse) Expression of negative regulators following DC-T cell co-culture eFluor-resting CD4+ T cells Baseline PD-1/Tim-3/CTLA-4 Phenotyping on days 1, 2, 3, 4, 5 post-infection eFluorhiEGFP- Viable Single cells T cells PD-1, Tim-3 and CTLA-4 SSC-W eFluor SSC-A CD3 FSC-A SSC-H HLA-DR EGFP

  9. Expression of negative regulators following DC-T cell co-culture PD-1 Tim-3 ( ) Mean fold change 24 Mean fold change 32 % Positive cells n=3 Days post-infection Days post-infection CTLA-4 No change % Positive cells Days post infection

  10. Differential expression of negative regulator ligands on mDC and pDC Leitner PLoS Pathog 2013

  11. Latent infection PD-1 lo/- cells Are PD-1hi cells enriched for latency? PD-1 hi cells eFluorhiEGFP- CD4+ T cells Anti-PD-1 aCD3/28 +L8 eFluor670 EGFP

  12. HIV latency is enriched in PD-1hi cells

  13. Tim-3 lo/- cells HIV latency is enriched in Tim3hi cells Tim-3 hi cells eFluorhiEGFP- CD4+ T cells Anti-Tim-3

  14. Summary • Myeloid DC facilitate establishment of HIV latency in resting memory CD4+ T cells via direct infection • Negative regulators PD-1 and Tim-3 but not CTLA-4 are up-regulated on resting CD4+ T cells upon co-culture with mDC • Negative regulator ligands are differentially expressed by mDC and pDC • mDC-induced HIV latency is enriched in PD-1hi and Tim-3hi cells

  15. EGFP+ productively infected CD4+ T cells Latent Infection in sorted eFluorhiEGFP- CD4+ T cells Blocking interactions between negative regulators and their ligands Cell death virus production immune clearance PD-1/-L1blockade PD-1 PD-L1

  16. Implications • mDC may facilitate ongoing latent infection of resting CD4+ T cells leading to replenishment of the reservoir • Disrupting the function of PD-1 and/or Tim-3 could potentially be exploited to inhibit replenishment of the reservoir and/or reverse latency

  17. Acknowledgements Monash University • Sharon Lewin • Paul Cameron • Renée van der Sluis • Nitasha Kumar • Suha Saleh • Candida da Fonseca AMREP Flow Cytometry • Geza Paukovicks • Michael Thompson • Jeanne Le Masurier • Phil Donaldson VGTI Florida • Rafick Sekaly • Nicolas Chomont • Remi Fromentin University of Melbourne • Damian Purcell

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